The delivery of gene products, such as proteins and RNA, to animals or animal cells is desirable for a variety of applications. Such applications include treatment of infectious diseases, therapy of acquired or inherited diseases or conditions, induction of an immune response to a protein antigen, the study of various cellular functions, etc. A range of bacteria have therefore been developed and used for the delivery of therapeutic molecules.
For example, live, attenuated, pathogenic Gram negative bacteria have been developed as vaccines (Garmory et al., 2003). Furthermore, strategies have been developed to use live, attenuated, pathogenic Gram negative bacterial vaccine strains as vectors to deliver a variety of protective vaccine antigens via the mucosal route (Medina, 2001; Roland et al., 2005). Such strains are generally non-colonising.
Gram positive, non-pathogenic lactic acid bacteria are also currently being developed as oral live vaccines (Bermudez-Humaran et al., 2003). In contrast to the attenuated, pathogenic Gram negative bacterial strains, Gram positive bacteria, such as Lactococcus lactis, are non-pathogenic. Furthermore, the Gram positive bacterial vectors often have a limited ability to colonise. In addition to delivering vaccine antigens, L. lactis is also being developed for the delivery of a number of cytokines such as IL-10 (Steidler, 2002; Steidler et al., 2003; Huybhebaert et al., 2005), IL-12 (Bermudez-Humaran et al., 2003; Kimoto et al., 2004) and IL-2 (Steidler, 2002). Methods for the delivery of surface anchored proteins by Gram positive bacteria are disclosed in U.S. Pat. No. 6,737,521, and methods for the delivery of cytokines in L. lactis are described in U.S. Pat. No. 6,605,286.
Live bacterial vectors are also potentially useful in the treatment of infectious diseases. For example, live bacterial vectors are potentially useful in the treatment of infectious diseases such as necrotic enteritis. Necrotic enteritis is an enterotoxemic disease caused by Clostridium perfringens which leads to the development of necrotic lesions in the gut wall resulting in morbidity and mortality of poultry. It is also a multifactorial disease with complex and partly unknown epidemiology and pathogenesis (Kaldhusdal, 1999). The bacterium, C. perfringens is commonly found in the gastrointestinal tract of poultry (Tschirdewahn et al. 1991), the occurrence of necrotic enteritis, however, is sporadic (Cowen et al., 1987). Nevertheless, feed contaminated with C. perfringens has been implicated in outbreaks of necrotic enteritis in chickens (Kaldhusdal, 1999). Studies have also shown that healthy chickens have a relatively low number of C. perfringens in their gastrointestinal tracts, while an increase in the concentration of the bacteria can result in a necrotic enteritis condition (Craven et al., 1999).
Bacitracin, linocomycin and other antibiotics have been commonly used to treat poultry suffering from necrotic enteritis (Craven et al., 1999). However, due to the isolation of antibiotic-resistant strains of C. perfringens from chickens and turkeys (Devriese et al., 1993; Kondo, 1988; Watkins et al., 1997) and the general desire to reduce antibiotic use because of the potential link to antibiotic resistance in human pathogens, poultry health authorities and producers are increasingly interested in the development and application of new products to replace traditional antibiotics. To date, however, there have been no reports of the treatment of necrotic enteritis using live rationally designed bacterial vectors delivering therapeutic products. However, there have been reports of the use of probiotic strains of bacteria and the use of largely uncharacterised single isolates and mixed populations of bacteria for competitive exclusion treatments.
Despite having great potential, the live bacterial vectors used to date, in other systems, have substantial limitations. For example, when given orally, bacterial cells are exposed to harsh gastrointestinal conditions, resulting in short survival time and requiring that a large dose be given (Prakash and Jones, 2005). A consequence of clearance of the bacteria from a subject is that delivery of the biologically active protein is of limited duration. An additional problem associated with the use of attenuated Gram negative bacterial vectors has been the difficulty in obtaining a balance between residual virulence of invasive and/or reactogenic vectors (Tacket et al., 1992) and the effective delivery of the bioactive polypeptide, such as a immunogenic construct (Zhu et al., 2006).
Accordingly, there is a need for improved bacterial vectors that are not hindered by these limitations.